ABSTRACT: FGFR2 is recurrently amplified in 5% of gastric cancers and 1%-4% of breast cancers; however, this molecular alteration has never been reported in a primary colorectal cancer specimen. Preclinical studies indicate that several FGFR tyrosine-kinase inhibitors (TKIs), such as AZD4547, have in vitro activity against the FGFR2-amplified colorectal cell line, NCI-H716. The efficacy of these inhibitors is currently under investigation in clinical trials for breast and gastric cancer. Thus, better characterizing colorectal tumors for FGFR2 amplification could identify a subset of patients who may benefit from FGFR TKI therapies. Here, we describe a novel FGFR2 amplification identified by clinical next-generation sequencing in a primary colorectal cancer. Further characterization of the tumor by immunohistochemistry showed neuroendocrine differentiation, similar to the reported properties of the NCI-H716 cell line. These findings demonstrate that the spectrum of potentially clinically actionable mutations detected by targeted clinical sequencing panels is not limited to only single-nucleotide polymorphisms and insertions/deletions but also to copy-number alterations.
Project description:Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating "addiction" of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.
Project description:Although regorafenib has demonstrated survival benefits in patients with metastatic colorectal and gastrointestinal stromal tumors, no proven biomarker has been identified for predicting sensitivity to regorafenib. Here, we investigated preclinical activity of regorafenib in gastric and colorectal cancer cells to identify genetic alterations associated with sensitivity to regorafenib. Mutation profiles and copy number assays of regorafenib target molecules indicated that amplification of fibroblast growth factor receptor 2 (FGFR2) was the only genetic alteration associated with in vitro sensitivity to regorafenib. Regorafenib effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules in a dose-dependent manner and selectively in FGFR2-amplified cells. Regorafenib induced G1 arrest (SNU-16, KATO-III) and apoptosis (NCI-H716); however, no significant changes were seen in cell lines without FGFR2 amplification. In SNU-16 mice xenografts, regorafenib significantly inhibited tumor growth, proliferation, and FGFR signaling compared to treatment with control vehicle. Regorafenib effectively abrogates activated FGFR2 signaling in FGFR2-amplified gastric and colorectal cancer and, therefore, might be considered for integration into treatment in patients with FGFR2-amplified gastric and colorectal cancers.
Project description:BACKGROUND:FGFR2 amplification is associated with aggressive gastric cancer (GC), and targeted drugs have been developed for treatment of GC. We evaluated the antitumor activity of an FGFR inhibitor in FGFR2-amplified GC patients with peritoneal carcinomatosis. METHODS:Two GC patients with FGFR2 amplification confirmed by fluorescence in situ hybridization showed peritoneal seeding and malignant ascites. We used the patient-derived xenograft model; patient-derived cells (PDCs) from malignant ascites were used to assess FGFR2 expression and its downstream pathway using immunofluorescence analysis and immunoblot assay in vitro. Apoptosis and cell cycle arrest after treatment of FGFR inhibitor were analyzed by Annexin V-FITC assay and cell cycle analysis. RESULTS:FGFR2 amplification was verified in both PDC lines. AZD4547 as an FGFR inhibitor decreased proliferation of PDCs, and the IC50 value was estimated to be 250 nM in PDC#1 and 210 nM in PDC#2. FGFR inhibitor also significantly decreased levels of phosphorylated FGFR2 and downstream signaling molecules in FGFR2-amplified PDC lines. In cell cycle analysis, apoptosis was significantly increased in AZD4547-treated cells compared with nontreated cells. The proportion of cells in the sub-G1 stage was significantly higher in AZD4547-treated PDCs than in control cells. CONCLUSION:Our findings suggest that FGFR2 amplification is a relevant therapeutic target in GC with peritoneal carcinomatosis.
Project description:BACKGROUND:Fibroblast growth factor 2 (FGFR2) amplification, occurring in ~2-9% of gastric cancers (GC), is associated with poor overall survival. RESULTS:RNA sequencing identified a novel FGFR2-ACSL5 fusion in the resistant tumor that was absent from the matched pre-treatment tumor. The FGFR2-amplified PDC line was sensitive to FGFR inhibitors whereas the PDC line with concomitant FGFR2 amplification and FGFR2-ACSL5 fusion exhibited resistance. Additionally, the FGFR2-amplified GC PDC line, which was initially sensitive to FGFR2 inhibitors, subsequently also developed resistance. MATERIALS AND METHODS:We identified an FGFR2-amplified patient with GC, who demonstrated a dramatic and long-term response to LY2874455, a pan-FGFR inhibitor, but eventually developed an acquired LY2874455 resistance. Following resistance development, an endoscopic biopsy was performed for transcriptome sequencing and patient-derived tumor cell line (PDC) establishment to elucidate the underlying molecular alterations. CONCLUSIONS:FGFR inhibitors may function against FGFR2-amplified GC, and a novel FGFR2-ACSL5 fusion identified by transcriptomic characterization may underlie clinically acquired resistance. IMPLICATIONS FOR PRACTICE:Poor treatment response represents a substantial concern in patients with gastric cancer carrying multiple FGFR2 gene copies. Here, we show the utility of a general FGFR inhibitor for initial response prior to treatment resistance and report the first characterization of a potential resistance mechanism involving an FGFR2-ACSL5 fusion protein.
Project description:Some gastric cancers have FGFR2 amplifications, making them sensitive to FGFR inhibitors. However, cancer cells inevitably develop resistance despite initial response. The underlying resistance mechanism to FGFR inhibition is unclear. In this study, we applied a kinome-wide CRISPR/Cas9 screen to systematically identify kinases that are determinants of sensitivity to a potent FGFR inhibitor AZD4547 in KatoIII cells, a gastric cancer cell line with FGFR2 amplification. In total, we identified 20 kinases, involved in ILK, SRC, and EGFR signaling pathways, as determinants that alter cell sensitivity to FGFR inhibition. We functionally validated the top negatively selected and positively selected kinases, ILK and CSK, from the CRISPR/Cas9 screen using RNA interference. We observed synergistic effects on KatoIII cells as well as three additional gastric cancer cell lines with FGFR2 amplification when AZD4547 was combined with small molecular inhibitors Cpd22 and lapatinib targeting ILK and EGFR/HER2, respectively. Furthermore, we demonstrated that GSK3b is one of the downstream effectors of ILK upon FGFR inhibition. In summary, our study systematically evaluated the kinases and associated signaling pathways modulating cell response to FGFR inhibition, and for the first time, demonstrated that targeting ILK would enhance the effectiveness of AZD4547 treatment of gastric tumors with amplifications of FGFR2.
Project description:Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.
Project description:<h4>Background</h4>Up to 10 % of primary gastric cancers are characterized by FGFR2 amplification, and fibroblast growth factor receptor (FGFR) inhibitors may represent therapeutic agents for patients with these malignancies. However, long-term benefits of the treatment might be limited owing to the occurrence of drug resistance.<h4>Methods</h4>To investigate the mechanisms of resistance to selective FGFR inhibitors, we established three FGFR2-amplified SNU-16 gastric cancer cell lines resistant to AZD4547, BGJ398, and PD173074, respectively.<h4>Results</h4>The resistant cell lines (SNU-16R) demonstrated changes characteristic of epithelial-to-mesenchymal transition (EMT). In addition, they displayed loss of expression of FGFR2 and other tyrosine kinase receptors concurrent with activation of downstream signaling proteins and upregulation of the transforming growth factor ? (TGF-?) level. However, treatment of parental SNU-16 cells with TGF-?1 did not evoke EMT, and pharmacological inhibition of TGF-? receptor I was not sufficient to reverse EMT changes in the resistant cells. Finally, we showed that the SNU-16R cell lines were sensitive to the human epidermal growth factor receptor 2 inhibitor mubritinib and the heat shock protein 90 inhibitor AUY922.<h4>Conclusion</h4>In conclusion, we provide experimental evidence that EMT-mediated resistance might emerge in gastric cancer patients following treatment with FGFR inhibitors, and mubritinib or AUY922 treatment may be an alternative therapeutic strategy for these patients.
Project description:Fibroblast growth factor receptor 2 (FGFR2)-targeted therapy has attracted considerable attention as novel anticancer agents in gastric cancer (GC). However, intrinsic or acquired drug resistance has emerged as a major challenge to their clinical use. In this study, we demonstrated that several receptor tyrosine kinase (RTK), including EGFR, HER3 and MET, activations contributed to AZD4547 (a selective FGFR2 inhibitor) hyposensitivity in FGFR2 amplified GC cells. The rescue effect was abrogated by inhibiting these RTKs with their targeted tyrosine kinase inhibitors (TKIs). In addition, synergy in growth inhibition was observed when the GC cells were treated with a combination of AZD4547 and cetuximab (an EGFR monoclonal antibody) both in vitro and in vivo. More importantly, tissue microarray analysis revealed that these resistance-conferring RTKs were highly expressed in FGFR2 positive GC patients. Taken together, these observations demonstrated RTKs including EGFR, HER3 and MET activations as novel mechanisms of hyposensitivity to AZD4547. It will be clinically valuable to investigate the involvement of RTK-mediated signaling in intrinsicor acquired resistance to FGFR2 TKIs in GC. A combination targeted therapeutic strategy may be recommended for treating FGFR2 amplified GC patients with these RTK activations.
Project description:Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers (e.g. liver, gastric, ovarian, endometrial, and bladder). ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent in vitro and in vivo activity against FGFR addicted cell lines and tumors. Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway (FRS2?, AKT, ERK) was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).